PROJECT SUMMARY Aedes aegypti transmits several arboviral diseases, including dengue and Zika fever, which threaten virtually half of the world?s population. Two subspecies, Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf), have been described based on their body coloration. These two subspecies differ remarkably from each other in their worldwide distribution, association with humans, and ability to transmit pathogens. In Anopheles populations, polymorphic inversions are often responsible for epidemiologically important phenotypes but our knowledge about chromosomal rearrangements in Aedes populations is scarce. So far, only two chromosomal inversions have been directly observed in chromosomes of Ae. aegypti from Senegal. Based on our preliminary data, we hypothesize that chromosomal inversions are abundant in Ae. aegypti and are involved in the establishment and maintenance of genomic and phenotypic divergence in natural populations of this mosquito. In this study, we will take advantage of a dramatically improved, fully re-annotated genome assembly for Ae. aegypti and employ the Hi-C approach along with Oxford Nanopore Technology (ONT) sequencing to characterize chromosomal rearrangements. [The primary goal of this R21 proposal is to identify chromosomal rearrangements in aedine mosquitoes]. Toward this end, we propose three specific aims: 1) characterize chromosomal rearrangements in 16 strains of Aaa and Aaf from various worldwide populations using the Hi-C approach; 2) develop a high-quality de novo genome assembly for the Aaf Uganda strain using advanced genome technologies; and 3) develop PCR and FISH-based approaches for identification of the 3p2 chromosomal inversion, which is potentially of medical importance. The innovative strategies of using Hi-C analysis and ONT sequencing will make possible the discovery of chromosomal inversions in Ae. aegypti and will stimulate future genetic studies aimed at preventing mosquito-borne disease transmission.